Endocannabinoids are implicated in the infarct size-reducing effect conferred by heat stress preconditioning in isolated rat hearts

OBJECTIVE: We have investigated the involvement of the endocannabinoid system in the delayed cardioprotection conferred by heat stress preconditioning in the isolated rat heart. METHODS: Rats were divided into eight groups (n=7 in each group), subjected to either heat stress (42 degrees C for 15 min, HS groups) or sham anaesthesia (Sham groups). Twenty-four hours later, their hearts were isolated, retrogradely perfused, and subjected to a 30-min occlusion of the left coronary artery followed by 120 min of reperfusion. Some hearts were perfused with either SR 141716 (a cannabinoid CB(1) receptor antagonist, 1 microM), SR 144528 (a CB(2) receptor antagonist, 1 microM) or L-NAME (a NOS inhibitor, 3 microM) 5 min before ischaemia and during the ischaemic period. RESULTS: The infarct size-reducing effect conferred by heat stress (35.7+/-1.8% in Sham to 14.1+/-0.6% in HS groups) was not altered by the perfusion of SR 141716 (11.2+/-1.5%) but was abolished by both SR 144528 (36.6+/-1.6%) and L-NAME (32.0+/-4.4%). In hearts from non-heat-stressed rats, perfusion with SR 141716 (32.8+/-1.6%), SR 144528 (33.4+/-2.2%) and L-NAME (31.6+/-2.9%) had no effect on infarct size. CONCLUSION: These results suggest an involvement of endocannabinoids, acting through CB(2) receptors, and NO in the cardioprotection conferred by heat stress against myocardial ischaemia. The possible interaction between both mediators of the heat stress response remains to be determined.     

Myocardial effects of ETB receptor stimulation.

INTRODUCTION: Endothelin-1 (ET-1) is an endogenous peptide whose effects are mediated by two distinct types of receptors, ETA and ETB. Whereas the ETA receptors promote vasoconstriction and mitogenesis and increase inotropism, ETB receptors have vasodilatory and anti-mitogenic properties mediated by nitric oxide release. In this study we investigated the myocardial effects of selective ETB receptor stimulation. METHODS: The study was performed on right papillary muscles (n = 30) from New Zealand white rabbits (Krebs-Ringer; 1.8 mM CaCl2; 35 degrees C). The effects of selective ETB receptor activation by Sarafotoxin S6c (0.2 microM; n = 6) and of non-selective ETA and ETB receptor activation by ET-1 (1 nM; n = 9) were studied. The effects of ET-1 were also evaluated in the presence of a selective ETA receptor antagonist, BQ-123 (0.1 microM; n = 9) and of a selective ETB receptor antagonist BQ-788 (0.1 microM; n = 6). Only significant results (mean +/- SE; p < 0.05) are given, expressed as % baseline. RESULTS: Sarafotoxin S6c reduced active tension (AT) by 8.1 +/- 5.5% and peak rate of tension development (dT/dtmax) by 8.6 +/- 5.6%. Alone, ET-1 increased AT by 64.2 +/- 18.2% and dT/dtmax by 58.6 +/- 20.2%. These effects of ET-1 were exacerbated in the presence of BQ-788 (AT increased by 82.6 +/- 17.5% and dT/dtmax by 121.3 +/- 26.6%) and inverted in the presence of BQ-123 (AT decreased by 12.8 +/- 2.7% and dT/dtmax by 16.1 +/- 3.0%). CONCLUSIONS: This study demonstrates, for the first time, that selective stimulation of ETB receptors has a negative inotropic effect. Detailed characterization of the effects of stimulation of each type of ET-1 receptor is of particular relevance as selective and non-selective inhibitors of these receptors are currently being tested for treatment of heart failure.     

Role of endothelin-1/endothelin receptor system in endotoxic shock rats.

Endothelin (ET)-1, a potent vasoconstrictor peptide derived from the endothelium, is markedly increased in endotoxic shock, although the pathophysiological role of ET-1 under septic conditions remains obscure. To delineate the role of ET-1 and its receptor subtype in endotoxic shock, we here attempted to determine the changes of circulating levels of ET-1 and its biosynthetic intermediate big ET-1 in endotoxic shock rats, to evaluate the gene expression of ET-1 as well as the ET-1 receptor subtypes (ETA and ETB) in the heart, lung and liver, and to study the effects of ET receptor antagonists on systemic arterial blood pressure, heart rate and survival rate. Administration of bacterial lipopolysaccharide (LPS) caused profound hypotension, increased heart rate and death, and these effects were blocked by a nonselective ETA/ETB receptor antagonist (TAK044), but not by an ETA selective antagonist (BQ123). Administration of exogenous ET-1 caused a profound pressor response in control rats, but not in the LPS-pretreated rats. Injection of LPS caused marked elevation of plasma levels of both ET-1 and big ET-1, which were not affected by treatment with either ET receptor antagonist. Administration of LPS caused up-regulation of ET-1 and ETB receptor mRNA in the heart, whereas ETA receptor mRNA was markedly down-regulated in the heart, lung and liver. These data suggest differential gene regulation of ET-1 and its receptor subtypes in various organs from endotoxic shock rats, and that nonselective ETA/ETB receptor antagonist, but not ETA receptor antagonist, ameliorates endotoxin-induced hypotension and death.     

Cardiovascular effects of 2-arachidonoyl glycerol in anesthetized mice

Cannabinoids, including the endogenous ligand anandamide, elicit pronounced hypotension and bradycardia through the activation of CB1 cannabinoid receptors. A second endogenous cannabinoid, 2-arachidonoyl glycerol (2-AG), has been proposed to be the natural ligand of CB1 receptors. In the present study, we examined the effects of 2-AG on mean arterial pressure and heart rate in anesthetized mice and assessed the role of CB1 receptors through the use of selective cannabinoid receptor antagonists and CB1 receptor knockout (CB1(-/-)) mice. In control ICR mice, intravenous injections of 2-AG or its isomer 1-AG elicit dose-dependent hypotension and moderate tachycardia that are unaffected by the CB1 receptor antagonist SR141716A. The same dose of SR141716A (6 nmol/g IV) completely blocks the hypotensive effect and attenuates the bradycardic effect of anandamide. 2-AG elicits a similar hypotensive effect, resistant to blockade by either SR141716A or the CB2 antagonist SR144528, in both CB1(-/-) mice and their homozygous (CB1(+/+)) control littermates. In ICR mice, arachidonic acid (AA, 15 nmol/g IV) elicits hypotension and tachycardia, and indomethacin (14 nmol/g IV) inhibits the hypotensive effect of both AA and 2-AG. Synthetic 2-AG incubated with mouse blood is rapidly (<2 minutes) and completely degraded with the parallel appearance of AA, whereas anandamide is stable under the same conditions. A metabolically stable ether analogue of 2-AG causes prolonged hypotension and bradycardia in ICR mice, and both effects are completely blocked by SR141716A, whereas the same dose of 2-AG-ether does not influence blood pressure and heart rate in CB1(-/-) mice. These findings are interpreted to indicate that exogenous 2-AG is rapidly degraded in mouse blood, probably by a lipase, which masks its ability to interact with CB1 receptors. Although the observed cardiovascular effects of 2-AG probably are produced by an arachidonate metabolite through a noncannabinoid mechanism, the CB1 receptor-mediated cardiovascular effects of a stable analogue of 2-AG leaves open the possibility that endogenous 2-AG may elicit cardiovascular effects through CB1 receptors.     

Endothelin receptor blockade improves endothelial function in atherosclerotic patients on angiotensin converting enzyme inhibition

OBJECTIVES: Endothelin-1 (ET-1) and angiotensin II may contribute to endothelial dysfunction, which is associated with increased risk of events in patients with coronary artery disease. The objective was to test whether dual ETA/ETB receptor antagonism improves endothelium-dependent vasodilatation (EDV) in atherosclerotic patients, also on treatment with angiotensin converting enzyme (ACE) inhibitor. DESIGN AND SETTING: EDV and endothelium-independent vasodilatation were determined in 37 patients with atherosclerosis during measurement of forearm blood flow (FBF) with venous occlusion plethysmography. The patients were then randomized to treatment with ramipril 10 mg o.d. (n=21) or placebo (n=16) for 3 months in a double-blind fashion. RESULTS: Intra-arterial infusion of the ETA receptor antagonist BQ123 and the ETB receptor antagonist BQ788 (both 10 nmol min(-1)) increased basal FBF by 42 +/- 4% (P <0.001) and enhanced EDV (P <0.001). Following 3 months ramipril treatment, ET receptor blockade still enhanced EDV. Acetylcholine 10 and 30 mg min(-1) increased FBF by 68 +/- 12 and 64 +/- 12 mL min(-1)/1000 mL before vs. 101 +/- 17 and 101 +/- 16 mL min(-1)/1000 mL following ET receptor blockade in the ramipril group (P <0.001). CONCLUSIONS: Dual ETA/ETB receptor blockade improves endothelial function and exerts direct vasodilator effects in patients with atherosclerosis, also on treatment with ramipril suggesting that ET receptor blockade may have important therapeutic effects when added to ACE inhibition in these patients.     

Autocrine effects of endothelin-1 on leukocyte-endothelial interaction: stimulation of endothelin B receptor subtype reduces endothelial adhesiveness via a nitric oxide-dependent mechanism

The effects of endothelin-1 (ET-1) on P-selectin-mediated leukocyte endothelial interaction were examined in vitro. Adherence of autologous polymorphonuclear leukocytes (PMNs) to the endothelium was markedly enhanced by endothelial stimulation with either (2 U/mL) thrombin, (1 mumol/L) histamine, or (100 nmol/L) phorbol myristate acetate (PMA). In contrast, ET-1 alone (10 and 100 nmol/L) only slightly increased the number of adhering PMNs. The increased PMN adherence to thrombin- or histamine-stimulated endothelium, which was blocked by an anti-P- selectin monoclonal antibody, was also significantly attenuated by preincubation of coronary segments with (100 nmol/L) ET-1. We further investigated the mechanism of this anti-adherence action of ET-1 on thrombin-stimulated endothelial adhesiveness. Preincubation of coronary segments with a selective ETA receptor antagonist, BQ485 (1 mumol/L), had no effect on ET-1 inhibition of thrombin-induced PMN adherence. In contrast, preincubation with a selective ETB receptor antagonist, BQ788 (1 mumol/L) significantly reversed ET-1 inhibition of thrombin-induced PMN adherence, whereas the selective ETB receptor agonist BQ-3020 mimicked the inhibitory action of ET-1 on thrombin-induced PMN adherence. Furthermore, (100 mumol/L) N omega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, significantly attenuated ET-1 inhibition of thrombin-stimulated PMN adherence. These results suggest that ET-1 may inhibit P-selectin-mediated leukocyte- endothelial interaction via ETB receptor stimulation and subsequent endothelial NO formation. This autocrine effect of ET-1 may be involved in pathophysiologic states such as early atherogenesis by preventing leukocyte-endothelial interaction in constricted blood vessels.     

Synthesis of platelet-activating factor and its receptor expression in Kupffer cells in rat carbon tetrachloride-induced cirrhosis

AIM： To determine the platelet-activating factor （PAF） synthesis and its receptor expression in Kupffer cells in rat carbon tetrachloride-induced cirrhosis.
METHODS： Kupffer cells, isolated from the livers of control and CCl4-induced cirrhotic rats, were placed in serum-free medium overnight. PAF saturation binding, ET-1 saturation and competition binding were assayed. ET-1 induced PAF synthesis, mRNA expression of PAF, preproendothelin-1, endothelin A （ETA） and endothelin B （ETB） receptors were also determined.
RESULTS： A two-fold increase of PAF synthesis （1.42 ± 0.14 vs 0.66 ± 0.04 pg/μg DNA） and a 1.48-fold increase of membrane-bound PAF （1.02 ± 0.06 vs 0.69 ± 0.07 pg/μg DNA） were observed in activated Kupffer cells of cirrhotic rats. The application of ET-1 to Kupffer cells induced PAF synthesis in a concentration-dependent manner in both cirrhotic and normal rats via ETB receptor, but PAF synthesis in the activated Kupffer cells was more effective than that in the normal Kupffer cells. In activated Kupffer cells, PAF receptor expression and PAF binding capacity were markedly enhanced. Activated Kupffer cells raised the [^125I]-ET-1 binding capacity, but changed neither the affinity of the receptors, nor the expression of ETA receptor.
CONCLUSION： Kupffer cells in the course of CCl4- induced cirrhosis are the main source of increased PAF. ET-1 is involved endogenously in stimulating the PAF synthesis in activated Kupffer cells via ETB receptor by paracrine. ETA receptor did not appear in activated Kupffer cells, which may exacerbate the hepatic and extrahepatic complications of cirrhosis.     

Endogenous cannabinoids mediate hypotension after experimental myocardial infarction

OBJECTIVES: We sought to determine whether endocannabinoids influence hemodynamic variables in experimental models of acute myocardial infarction (MI). BACKGROUND: Hypotension and cardiogenic shock are common complications in acute MI. Cannabinoids are strong vasodilators, and endocannabinoids are involved in hypotension in hemorrhagic and septic shock. METHODS: The early effect of left coronary artery ligation on hemodynamic variables was measured in rats pretreated with the selective cannabinoid(1) receptor (CB(1)) antagonist SR141716A (herein referred to as SR, 6.45 micromol/kg body weight intravenously) or vehicle. Endocannabinoids produced in monocytes and platelets were quantified by liquid chromatography/mass spectrometry (LC/MS), and their effects on blood pressure and vascular reactivity were determined. RESULTS: After MI, mean arterial pressure (MAP) dropped from 126 +/- 2 mm Hg to 76 +/- 3 mm Hg in control rats, whereas the decline in blood pressure was smaller (from 121 +/- 3 mm Hg to 108 +/- 7 mm Hg, p < 0.01) in rats pretreated with SR. SR increased the tachycardia that follows MI (change [Delta] in heart rate [HR] = 107 +/- 21 beats/min vs. 49 +/- 9 beats/min in control rats, p < 0.05). The MI sizes were the same in control rats and SR-treated rats. Circulating monocytes and platelets isolated 30 min after MI only decreased MAP when injected into untreated rats (DeltaMAP = -20 +/- 5 mm Hg), but not in SR-pretreated rats. The endocannabinoids anandamide and 2-arachidonyl glycerol were detected in monocytes and platelets isolated after MI, but not in cells from sham rats. Survival rates at 2 h after MI were 70% for control rats and 36% for SR-treated rats (p < 0.05). Endothelium-dependent arterial relaxation was attenuated in SR-treated rats (maximal relaxation: 44 +/- 3% [p < 0.01] vs. 70 +/- 3% in control rats) and further depressed by SR treatment (24 +/- 5%, p < 0.01 vs. MI placebo). CONCLUSIONS: Cannabinoids generated in monocytes and platelets contribute to hypotension in acute MI. Cannabinoid(1) receptor blockade restores MAP but increases 2-h mortality, possibly by impairing endothelial function.     

Altered endothelin-1 binding following balloon angioplasty of pig coronary arteries: effect of the ETA receptor antagonist, LU 135252.

OBJECTIVE: Since raised levels of endothelin-1 (ET-1) have been detected in the human coronary sinus following percutaneous transluminal angioplasty (PTCA) we investigated the role of ET-1 in the etiology of vascular restenosis. METHODS: Balloon angioplasty of coronary arteries was performed in pigs and the animals were treated with placebo or the endothelin (ETA) receptor antagonist LU 135252 (30 mg/kg/day). After 4 weeks vascular stenosis and the distribution of endothelin and its receptors was evaluated. RESULTS: The pronounced neointima formation in the control group (neointima:media ratio = 0.87 +/- 0.36) was significantly reduced by LU 135252 (0.43 +/- 0.30, P < 0.001). Angioplasty caused a significant increase in medial ETA (approximately 275%, P < 0.026) and ETB (approximately 250%, P < 0.001) binding to injured, compared with non-injured segments, an effect that was also reduced by LU 135252 (ETA = 11.5% increase; ETB = 14% increase). The neointima of control animals exhibited ET-1 like immunoreactivity as well as ETA and ETB binding sites. CONCLUSION: These data indicate that endothelin is locally-released from endothelial and vascular smooth muscle cells following angioplasty which binds to ETA and ETB receptor sites in the neointima and media. Since administration of the ETA antagonist LU 135252 markedly reduces neointima formation and medial ET binding, we conclude that vascular smooth muscle cell proliferation and subsequent neointima formation is mediated predominantly via ETA receptors. These data underscore the therapeutic potential of ETA antagonists in reducing the degree of restenosis following vascular injury.     

Functional role of ETB receptors in the renal medulla

Experiments were conducted to determine the influence of ETB receptors in the control of renal medullary function. The acute relation between renal perfusion pressure (RPP) and natriuresis was examined in anesthetized rats treated with the ETB antagonist A-192621 (10 mg/kg IV). In A-192621-treated rats, sodium excretion (UNaV) was 0.4+/-0.1, 0.6+/-0.3, and 2.7+/-0.5 micromol/min at RPP of 80+/-1, 107+/-1, and 144+/-5 mm Hg, respectively. In control rats, UNaV averaged 0.8+/-0.4, 3.4+/-1.2, and 8.1+/-1.7 micromol/min at RPP of 77+/-2, 115+/-5, and 137+/-3 mm Hg, respectively. For normal and high RPP, UNaV was significantly lower in A-192621-treated rats compared with control rats. Additional experiments determined the effects of Big ET-1 (10 pmol/kg per minute) on intrarenal blood flow. Medullary blood flow (MBF) and cortical blood flow were measured in anesthetized rats by single-fiber, laser Doppler flowmetry. Cortical blood flow significantly decreased in response to Big ET-1 in rats on a normal or high salt diet. Big ET-1 significantly increased MBF in rats on a high salt diet, whereas there was no change in MBF in rats on a normal salt diet. These results demonstrate that medullary vasodilation produced by Big ET-1 is more prominent in rats on a high salt diet and are consistent with a contribution of ETB-mediated events in the natriuretic response to high salt intake. Taken together, these findings support the hypothesis that endothelin plays an important role in regulating sodium excretion through activation of ETB receptors.     

Losartan-induced attenuation of blood pressure in L-NAME hypertensive rats is associated with reversal of the enhanced expression of Gi alpha proteins

OBJECTIVE: We have previously reported that hearts from N-[omega]-nitro-L-arginine methyl ester (L-NAME)-induced hypertensive rats exhibited an enhanced expression of Gi proteins. Since, losartan, an AT1 receptor antagonist, has been shown to attenuate the L-NAME-induced increase in blood pressure, we undertook the present studies to evaluate whether losartan-induced decreased blood pressure in this model of hypertension is associated with attenuation of enhanced expression of Gi proteins and adenylyl cyclase signalling. METHODS: L-NAME (70 mg/kg body weight) and losartan (10 mg/kg body weight), alone or in combination, were given orally to Sprague-Dawley rats for 4 weeks. The control rats received only plain tap water. The levels of inhibitory guanine nucleotide regulatory proteins (Gi alpha-2 and Gi alpha-3) and stimulatory (Gs alpha) proteins and Gi alpha mRNA in hearts were determined by immunoblotting and Northern blotting, respectively. Adenylyl cyclase activity was determined by measuring [32P]cAMP formation from [32P]ATP. RESULTS: Systolic blood pressure was enhanced in L-NAME-treated rats compared to control rats (164 +/- 5.2 versus 105 +/- 2 mmHg; n = 30), and was significantly attenuated by losartan treatment (164 +/- 5.2 mmHg versus 120 +/- 2.5 mmHg; n = 30). The expression of Gi alpha-2 and Gi alpha-3 proteins and their mRNA, which was enhanced in L-NAME-treated rats, was reversed by losartan treatment. However, losartan alone did not alter the levels of Gs alpha or Gi alpha proteins. In addition, the stimulatory effects of guanosine 5'-gamma-thiotriphosphate (GTPgammaS), isoproterenol, 5'-N-ethylcarboxamideadenosine (NECA), glucagon, forskolin (FSK) and sodium fluoride (NaF) on adenylyl cyclase, which were diminished in L-NAME-treated rats, were reversed by losartan treatment. Furthermore, the inhibition of forskolin-stimulated enzyme activity by low concentrations of GTPgammaS (receptor-independent Gi functions), which was significantly enhanced in L-NAME-treated rats, was attenuated by losartan treatment. In addition, losartan was able to reverse the attenuated receptor-mediated inhibitions of adenylyl cyclase by oxotremorine and angiotensin II towards control. CONCLUSIONS: These results suggest the implication of AT1 receptors in enhanced expression of Gi alpha proteins and increased blood pressure in L-NAME-induced hypertension.     

Combined endothelin receptor blockade evokes enhanced vasodilatation in patients with atherosclerosis

Endothelin (ET)-1 causes vasoconstriction via ET(A) and ET(B) receptors located on vascular smooth muscle cells and vasodilatation via ET(B) receptors on endothelial cells. Studies in vitro indicate an upregulation of ET(B) receptors in atherosclerosis. The present study investigated the vascular effects evoked by endogenous ET-1 in atherosclerotic patients. Forearm blood flow (FBF) was measured with venous occlusion plethysmography in 10 patients with atherosclerosis and in 10 healthy control subjects during intra-arterial infusion of selective ET receptor antagonists. The ET(B) receptor antagonist BQ788 evoked a significant increase in FBF (31+/-13%) in the patients, whereas a 20+/-9% reduction was observed in the control subjects. The ET(A) receptor antagonist BQ123 combined with BQ788 evoked a marked increase in FBF (102+/-25%) in the patients compared with no effect in the control subjects (-3+/-9%, P<0.001 versus patients). The ET(A) receptor antagonist BQ123 increased FBF to a similar degree in patients (39+/-11%) as in control subjects (41+/-11%). The increase in FBF evoked by selective ET(A) receptor blockade was significantly (P<0.05) less than that evoked by combined ET(A)/ET(B) receptor blockade in the atherosclerotic patients. These observations suggest an enhanced ET-1-mediated vascular tone in atherosclerotic patients, which is at least partly due to increased ET(B)-mediated vasoconstriction.     

Possible mechanism of endothelin-induced Ca2+ mobility in A7r5 cultured vascular smooth muscle cells

We studied the mechanism by which endothelin-1 (ET-1) affects the mobility of intracellular free Ca2+ ([Ca2+]i) in cultured A7r5 aortic smooth muscle cells. ET-1 at 10(-9) to 10(-7) M increased [Ca2+]i in Ca2+-containing buffer and Ca2+-free buffer. Pretreatment with ET-1 inhibited thapsigargin- and carbonylcyanide m-chlorophenylhydrazone (CCCP)-induced [Ca2+]i increases in Ca2+-free buffer. Pretreatment with thapsigargin and CCCP partially abolished the [Ca2+]i increase induced by ET-1. The ET-1-induced Ca2+ signal was partially suppressed by the ETA receptor antagonist BQ123 and the ETB receptor antagonist BQ788 and nifedipine. Pretreatment of cells with the phospholipase C inhibitor U73122 reduced the ET-1-induced [Ca2+]i increase. These results suggest that the ET-1-induced [Ca2+]i increase in A7r5 smooth muscle cells initially activates the ETA receptor, leading to Ca2+ influx and increased internal Ca2+ release from endoplasmic reticulum and mitochondrial Ca2+ stores. The ETB receptor and L-type Ca2+ channel are involved in maintaining further extracellular Ca2+ influx. ET-1-induced intracellular Ca2+ release was also modulated by phospholipase C-coupled events.     

Peroxisome proliferator-activated receptor-alpha activation reduces salt-dependent hypertension during chronic endothelin B receptor blockade

Endothelin B (ETB) receptor stimulation inhibits sodium transport in a similar fashion as 20-HETE. Clofibrate, a peroxisome proliferator-activated receptor-alpha (PPAR-alpha) agonist, increases protein expression of cytochrome P450 4A (CYP4A), which is responsible for 20-HETE synthesis in the kidney. Experiments were designed to determine whether clofibrate reduces hypertension associated with chronic ETB receptor blockade. Male Sprague-Dawley rats received either normal-salt (0.8% NaCl) or high-salt (8% NaCl) diet for 10 days. Female rats were fed a high-salt (8% NaCl) diet for 10 days. During the last 7 days, rats of both sexes were divided into 3 treatment groups: (1) clofibrate in drinking water (80 mg per day), (2) ETB receptor antagonist A-192621 in food (10 mg/kg per day), or (3) clofibrate and A-192621. During ETB receptor blockade, clofibrate had no effect on mean arterial pressure (MAP) under normal salt conditions. In contrast, clofibrate significantly inhibited the increase in MAP produced by A-192621 in rats fed a high-salt diet (34+/-3 versus 19+/-4 mm Hg; P <0.05). Similar results were observed in female rats administered A-192621 and fed a high-salt diet. ETB receptor blockade significantly decreased CYP4A protein expression in the renal cortex of rats on high salt. Clofibrate significantly increased renal cortical and medullary CYP4A protein expression in A-192621-treated male rats on high salt. Therefore, chronic PPAR-alpha agonist treatment reduces salt-dependent hypertension produced by ETB receptor blockade in male and female Sprague-Dawley rats. This suggests a possible relationship between ETB receptor activation and the maintenance of CYP4A protein expression in the kidney of rats fed a high-salt diet.     

Effects of CB1 cannabinoid receptor blockade on ethanol preference after chronic ethanol administration

BACKGROUND: Chronic ethanol administration results in neurobiological alterations similar to those observed after chronic cannabinoid exposure. The purpose of this study was to investigate alcohol drinking and the withdrawal responses after pulmonary chronic alcoholization with intraperitoneal or oral administration of a cannabinoid CB1 receptor antagonist. METHODS: The cannabinoid receptor antagonist SR141716A, 1, 3 or 10 mg/kg/day intraperitoneally or orally, was administered to Wistar rats either during a 30-day chronic ethanol exposure or at the cessation of this procedure. Motility was recorded during 18 hr after the cessation of chronic alcoholization just before the beginning of the free-choice paradigm (water versus alcohol 10% v/v). RESULTS: A significant increase in ethanol preference was observed during the free-choice paradigm after chronic alcoholization with concurrent SR141716A administration (3 or 10 mg/kg/day). A significant decrease in withdrawal motility after administration of SR141716A was observed with only the highest dose (10 mg/kg/day). The administration of SR141716A, 3 or 10 mg/kg/day, after chronic pulmonary alcoholization significantly decreased the preference for alcohol. Finally, a significant decrease in ethanol preference was seen during the free-choice paradigm of nonalcoholized rats treated with SR141716A, 3 or 10 mg/kg/day, during 30 days before the free-choice paradigm. CONCLUSIONS: The concurrent administration of the CB1 antagonist together with the chronic alcoholization increases the preference for ethanol. Also, the administration of the CB1 antagonist after the chronic alcoholization or at the time of withdrawal drastically diminishes the ethanol preference.     

Mechanisms underlying endothelin-1 effects on myocardial function]

INTRODUCTION: Endothelin-1 (ET-1) has potent vasoconstrictor, growth promoting and positive inotropic properties. Its effects on the intrinsic properties of the myocardium were recently described. The present study investigated the mechanisms underlying those effects. METHODS: The myocardial effects of 1 and 10 nM of ET-1 were evaluated in isolated rabbit papillary muscles (n = 9) and human atrial trabecula from CABG patients (Krebs-Ringer; 1.8 mM CaCl2; 35 degrees C). In papillary muscles the effects of 1 nM ET-1 were also studied in the presence of: (i) a selective ETA receptor antagonist, BQ-123 (0.1 microM; n = 9); (ii) a selective ETB receptor antagonist, BQ-788 (0.1 microM; n = 6); and (iii) an Na+/H+ exchanger inhibitor, methyl-isobutyl-amiloride (MIA; 1 microM; n = 6). Only significant results (mean +/- SE, p < 0.05) are given, expressed as delta % baseline. RESULTS: In AT by papillary muscles, 1 nM of ET-1 increased 64 +/- 16%, dT/dtmin 39 +/- 13% and decreased PT by 11 +/- 2%. The analysis of atrial strip contractions yielded similar results. In papillary muscles the effects of ET-1 were not affected by BQ-788, yet they were abolished by BQ-123, and reduced by 44% by MIA. CONCLUSIONS: The action of ET-1 on myocardial function is similar in human and non-human myocardium. The myocardial effects observed in the present study are mediated by the binding to ETA receptors, and partially dependent on Na+/H+ exchanger activation.     

Receptor- and non-receptor-mediated clearance of big-endothelin and endothelin-1: differential effects of acute and chronic ETA receptor blockade

AIMS: The aims of this study were to define and characterize the different mechanisms and sites of clearance of plasma endothelin-1 (ET-1) and big endothelin-1 (BigET-1) and evaluate possible effects of ETA versus combined ETA and ETB receptor blockade or endothelin converting enzyme (ECE) inhibition. METHODS: Time courses and sites of clearance were evaluated in Wistar-Kyoto rats after bolus injection of radiolabelled peptides into the carotid artery before or after treatment with LU1 35252 (ETA) and bosentan (ETA and ETB) as receptor antagonists or the ECE inhibitor phosphoramidon. RESULTS: The study shows that differential clearance of 125I-ET-1 and 125I-BigET-1 is mediated by distinct tissue-specific, receptor- and non-receptor-mediated mechanisms. Low levels of plasma ET-1 are rapidly cleared, mainly in the pulmonary circulation, through a low-capacity saturable ETB receptor-linked mechanism. In contrast, BigET-1 clearance is markedly slower, confined largely to liver and kidneys, is essentially non-receptor-mediated and is independent of converting enzyme activity. Acute inhibition of both ETA and ETB receptors with bosentan dramatically prolonged 125I-ET-1 plasma half-life and shifted tissue uptake from lung to liver and kidneys. Pulmonary clearance of 125I-ET-1 was decreased by chronic but not acute treatment with the specific ETA receptor antagonist LU135252. In contrast, 125I-Big-ET-1 clearance and tissue uptake were essentially unchanged by all treatments. CONCLUSIONS: Plasma levels and clearance studies on ET-1 and BigET-1 may provide differential information regarding pathological changes in their separate uptake mechanisms. Such data could have diagnostic or prognostic value in pulmonary, hepatic and renal pathophysiology or future therapeutic monitoring of treatment efficacy following administration of selective receptor antagonists.     

Renal endothelin receptor type B upregulation in rats with low or high renin hypertension

OBJECTIVES: To evaluate the role of endothelin-1 (ET-1) in hypertension, we investigated density and distribution of ETA and ETB receptors in hearts and kidneys of deoxycorticosterone acetate (DOCA)-salt and 1 kidney -- 1 clip (1K1C) hypertensive rats. METHODS: Five groups of uninephrectomized Wistar rats were put on a low salt diet. Three groups of rats drank tap water and two groups received saline. One group of each regimen received DOCA subcutaneously and two corresponding groups without DOCA served as controls. The fifth group of rats had the renal artery clipped to induce 1K1C hypertension. At 6 weeks, mean arterial pressure (MAP) was recorded and membrane binding assays using 125I-ET-1 were carried out. RESULTS: MAP was increased from control 122 +/- 3 to 155 +/- 6 and 218 +/- 11 mmHg in DOCA-salt and 1K1C rats, respectively, and cardiac weight index was increased. ETA receptors were predominantly expressed in the heart, whereas ETB receptors were predominant in the kidney. In the kidneys, the density of the ETB receptor subtype was upregulated in DOCA-salt and 1K1C rats from 160 +/- 8 to 217 +/- 12 and 190 +/- 2 fmol/mg (P < 0.05), respectively, and ETA tended to be downregulated (P = 0.057). Plasma renin activity was decreased in DOCA-salt rats from 17 +/- 3 to 0.17 +/- 0.01 ng/ml per h and increased in 1K1C rats on low salt diet to 30 +/- 5 ng/ml per h. CONCLUSIONS: Since ETB is the predominant endothelin receptor in the kidneys, upregulation of the ETB receptor mediating vasodilation and downregulation of the ETA receptor mediating vasoconstriction would be compatible with a mainly renal counter-regulatory effect of endothelin-1 to hypertension. Both low and high renin models of hypertension may be affected.     

Central administration of a specific angiotensin II receptor antagonist on baroreflex function in spontaneously hypertensive rats.

To determine whether the increase in baroreflex sensitivity previously reported in lifetime captopril-treated spontaneously hypertensive rats (SHR) was due to an inhibition of brain angiotensin II mechanisms, we tested the effect of intracerebroventricular administration of an angiotensin II receptor antagonist on baroreflex control of heart rate in lifetime captopril-treated and untreated male and female SHR. Baroreceptor reflex control of heart rate was assessed by the slope of the relationship between changes in mean arterial pressure (delta MAP, mmHg) and changes in pulse interval (delta PI, ms). MAP was raised and lowered with infusions of phenylephrine and nitroprusside, respectively. Following basal assessment of baroreflex control of heart rate, rats received an intracerebroventricular injection (100 pmol) of Sar1Thr8 angiotensin II (sarthran) and reflex control of heart rate was reassessed. Adequacy of blockade was tested with central and peripheral administration of angiotensin II. Captopril-treated male (130 +/- 7 mmHg) and female (123 +/- 4 mmHg) rats had significantly lower MAP than untreated rats (174 +/- 4 and 173 +/- 8 mmHg, respectively) and enhanced bradycardia in response to increases in MAP. Intracerebroventricular administration of sarthran had no effect on basal blood pressure or heart rate but enhanced the bradycardia in response to increases in MAP in both untreated and captopril-treated rats. The increase in the slope of the line relating delta MAP to delta PT was greater for untreated than captopril-treated SHR (male untreated 2.7-fold versus captopril-treated 1.5-fold; female untreated 1.6-fold versus captopril-treated 1.5-fold).(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelin B receptor-deficient mice develop endothelial dysfunction independently of salt loading

BACKGROUND: Rodents without a functional endothelin B (ETB) receptor develop salt-sensitive hypertension. The underlying mechanisms, however, are so far unknown. The ETB receptor is involved in endothelial function by modulating the activity of the endothelial nitric oxide synthesis as well as contributing to the control of endothelial prostacyclin synthesis. In the present study, we analysed whether salt alters endothelial function in rescued ETB receptor-deficient mice. We used mice with a rescue of the lethal phenotype of an ETB knockout. These mice were generated by crossbreeding ETB mice with dopamine-hydroxylase ETB transgenic mice. METHODS: Adult rescued ETB-deficient mice were kept in parallel with wild-type control animals for 15 days on standard (0.2% NaCl) or salt-enriched (4% NaCl) chow, respectively. Systolic blood pressure was measured by the tail cuff method and endothelium-dependent and endothelium-independent vascular function was assessed in isolated aortic rings under isometric conditions. RESULTS: Systolic blood pressure increased on salt-enriched chow in ETB receptor-deficient mice (166 +/- 12 mmHg), but neither in wild-type mice on high-salt diet (128 +/- 11 mmHg; P < 0.05) nor in ETB receptor-deficient mice on standard chow. The heart rate was similar in all groups at any point of time. Endothelium-dependent relaxation was impaired in ETB receptor-deficient mice (74 +/- 3 versus 96 +/- 5% of preconstriction for wild-type mice; P < 0.05) and was not significantly affected by a salt-enriched diet. Endothelium-independent relaxation was similar among all groups. Contractions to endothelin-1 were not significantly influenced by preincubation with the ETB receptor antagonist BQ-788, but were completely blunted by preincubation with the ETA receptor antagonist BQ-123 in all animals. CONCLUSION: Rescued ETB receptor-deficient mice develop salt-sensitive hypertension. Nevertheless, in this animal model of ETB receptor deficiency, endothelial function is impaired independent of salt-enriched diet or hypertension. This indicates that, in this model, salt-induced hypertension is not mediated by endothelial dysfunction.